A quantitative assessment of osteoinductivity of human demineralized bone matrix.

Demineralized bone matrix (DBM) is widely used in the repair of pathologies associated with skeletal defects and periodontal diseases. The present study was directed at establishing in vivo and in vitro models for a quantitative assessment of the osteoinductivity of DBM before clinical use. Athymic mice were used in an in vivo assay to overcome the species limitations (for human DBM) found in xenogeneic animal models. Calcium contents of explants, as an indicator of new bone formation, were assayed and expressed as a change in the weight percent calcium in the explant as compared to the weight percent of calcium in the implanted material. A total of 82 mice (2 implants per mouse) were used in this study. Significant amounts of new bone were induced in this animal model in response to implantation of DBM. Muscular implantation was found to be more osteoinductive (increases of 10.0 +/- 0.4 calcium weight percent of explant) than subcutaneous implantation (increases of 1.62 +/- 0.27 calcium weight percent of explant) and new bone formation in muscular implantation sites of athymic mice mimics endochondral bone formation. Between weeks 1 to 4, the weight of explanted materials did not significantly differ from the weight of the implanted material; however, by week 5 the explant weight began to increase. Calcium deposition over the 5 weeks of implantation increased in a nearly linear fashion. Consequently week 4 was chosen as the optimum time for explantation in the in vivo assay in that sufficient calcium levels had been achieved without a significant increase in explant dry weight. Aliquots of 10, 20, 30, and 40 mg per implantation site were used in dose response studies in the in vivo bioassay. Dose response curves with DBM exhibited maximal activity at the 20 mg DBM implant dose in the in vivo bioassay. An in vitro bioassay was also developed where human periosteal (HPO) cells were chosen because osteoprogenitor cells found in bone repair typically come from periosteal tissue. Alkaline phosphatase (ALP) activity in confluent cell cultures of HPO cells exposed to DBM, as an indicator of osteoblast induction, reached its highest level on day 5 of DBM treatment. Aliquots of 2, 5, 10, 20, 30, and 40 mg DBM per flask were chosen in dose response studies using the in vitro bioassay. These dose response studies with DBM revealed that quantities approximating 5 to 10 mg DBM in the in vitro model provided for maximal levels of ALP in cell extracts. A linear correlation (R2 = 0.7397) was demonstrated between the in vivo calcium remineralization assay and the in vitro ALP assay of osteoinductivity of DBM, suggesting that the in vitro assay can be used to quantitatively assess the osteoinductive potential of DBM where production and distribution of clinically usable DBM dictates rapid analysis.

Effect(s) of the demineralization process on the osteoinductivity of demineralized bone matrix.

The relationships between residual calcium levels and particle size of ground demineralized bone matrix and its osteoinductive potential were investigated using in vitro and in vivo assays. The effects of variable residual calcium levels, variable particle sizes, and donor age and gender were studied using a tissue culture-based bioassay (in vitro) as well as an athymic mouse (in vivo) bioassay. The osteoinductive potential of the bone-derived biomaterial was assessed by measuring the degree of new bone formation (change in percent calcium content after 4 weeks of implantation) in the in vivo assay and levels of alkaline phosphatase activity associated with cultures of human periosteal cells (HPO cells) in the in vitro assay, respectively. Slightly demineralized bone matrix and overly demineralized bone matrix possessed a degree of osteoinductive potential whereas bone demineralized to levels of approximately 2% residual calcium provided for maximum osteoinductive potential in both assay systems. The osteoinductive potential of ground demineralized bone varied relative to the particle size such that DBM particles ranging from 500 to 710 microns provided for the highest level of calcium deposition (increase of 8.1 weight percent calcium) after 4 weeks of implantation in muscle pouches of an athymic mouse, whereas explanted particles less than 250 microns showed the lowest level of calcium deposition (increase of only 2.8 weight percent calcium). In the donor age and gender study, DBM from different donors were divided into 5 age groups for both female and male donor derived bone: less than 20, 21 to 30, 31 to 40, 41 to 50, and 51 to 60 year old age groups. This study indicated that DBM from female donors in the 31 to 40 years old age group and male donors in the 41 to 50 year age group possess the highest osteoinductive potential, whereas DBM derived from donor bone from both female and male donors in the 51 to 60 year age group presented the lowest osteoinductive potential. DBM derived from male and female donors did not in general show significant differences in osteoinductive potential.

The incidence of osteo-arthritis of the temporomandibular joint in various cultures.

Three hundred and forty-eight cranial remains from Bronze and Iron Age British, Romano-British, Anglo-Saxon, Eastern Coast Australian aborigines, Medieval Christian Norse, Medieval Scarborough, 17--20th century British and German cultures, were examined for the presence of osteoarthritis in the temporomandibular joints. Cultures exposed to more stringent living conditions and with well-worn teeth had about twice the incidence of osteo-arthritis as the more sophisticated cultures. In general, loss of either molar support or occlusal imbalance were potent aetiological factors in this disease.

Variation of docosahexaenoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage.

The oxidation of phospholipid-bound docosahexaenoic acid (DHA) has been shown to be one of the major factors that limit the motile life span of sperm in vitro. Sperm samples show high cell-to-cell variability in life span and, consequently, in susceptibility toward lipid peroxidation. Therefore, we postulated that there is also cell-to-cell variability in DHA concentration in human spermatozoa. In this study, the concentration of DHA in subsets of human spermatozoa isolated by a discontinuous Percoll density gradient was determined by gas chromatography. Four subsets of human spermatozoa were isolated using a discontinuous Percoll gradient: fraction 1 was enriched in immature germ cells and immature sperm, fractions 2 and 3 contained, mostly, immature sperm with cytoplasmic droplets, and fraction 4 contained, for the most part, morphologically normal sperm, as determined by histochemical analysis. The results indicated that there were significant differences in DHA content in sperm from all 4 fractions. DHA content in sperm from fraction 1 was 2.5-fold higher than that found in fraction 4. DHA content in mouse sperm obtained from the seminiferous tubules was 3-fold higher than that found in mouse sperm obtained from the epididymis, consistent with the findings observed in ejaculated human sperm. The results of this study indicate (i) there is cell-to-cell variability in the concentration of DHA in human sperm and (ii) that there is a net decrease in DHA content in sperm during the process of sperm maturation.

Recruitment and discharge patterns of single motor units during speech production.

Recruitment and discharge patterns of single motor units (MUs) in the anterior belly of digastric were studied during speech in three subjects, using electrodes facilitating selective recording at high force levels. Fixed recruitment order was observed in over 99% of all comparisons. Later recruited units invariably possessed muscle action potentials of higher amplitude, suggesting that units were activated in accordance with the "size principle." Additional evidence for this was that later recruited units, of a set of three studied during speech, motor unit training, and isometric force ramps, showed greater sensitivity to input, and greater dynamic range than earlier recruited units. Units in this set were much more sensitive to rapid changes in input associated with speech gestures than to static activation even at high force levels. Several significant relations between discharge characteristics and aspects of movement dynamics were observed, including relations between (1) recruitment interval (MU1 to MU3) and latency of mandibular lowering, (2) onset of initial discharge of MU1 and relative mechanical advantage of the mandible, (3) number of MUs active and velocity and displacement of the mandible, and (4) discharge rate of MU3 and velocity and displacement of the mandible.

Mechanical properties of single motor units in speech musculature.

Electromyograms were recorded from three subjects during steady, low-rate discharge of single motor units of anterior belly of digastric, accompanying maintenance of isometric tension. The overall tension developed in muscles subserving mandibular depression was recorded from a force tansducer situated inferior to the mandibular symphysis. The force transducer output from the 90 ms period immediately following each of the several hundred discharges of a motor unit was averaged to obtain a characteristic tension curve for the contractile response (twitch) of that unit. Peak twitch tensions and time taken to develop peak tension (contraction time) were recorded for 27 motor units. The obtained median twitch tension of 0.26 g and contraction time of 29 ms were compared with values obtained for units in other human muscles and in animal muscles. As expected from the size principle, later recruited units possessed higher twitch tensions. Contrary to some statements of the size principle, contraction times were not shorter in later recruited units. Data on muscles elevating the mandible were also evaluated.

The oral health status of individuals on renal dialysis.

The primary purpose of this investigation was to determine the oral health status of individual's undergoing renal dialysis in southeastern Virginia. A sample was identified for this cross-sectional study via a local health maintenance organization. Three subgroups of the population were studied: 1) those who have been on renal dialysis for less than a year; 2) those on renal dialysis for 1 to 3 years; and 3) those on renal dialysis for longer than 3 years. Three dental indices the Periodontal Disease Index; the Decayed, Missing. Filled index; and the Simplified Oral Hygiene Index--were used to identify periodontal disease, dental caries activity, and oral hygiene status. Data were compiled and analyzed by using the parametric test, 1-way analysis of variance. Results suggested that 100% (n = 45) of the individuals undergoing renal dialysis presented with some form of periodontal disease (X = 3.15, SD = 1.41). The majority (64%) of the sample displayed either severe gingivitis (28%) or early periodontitis (36%). Sixty-four percent of the sample displayed a high DMF index (X = 11.77, SD = 7.55), while 98% of the sample accumulated calculus. Oral debris was present in 100% of the sample, resulting in a high Simplified Oral Hygiene Index score (X = 3.24, SD = 1.26), suggesting an increased need for oral care instruction. Findings led to the conclusion that the renal dialysis population in southeastern Virginia, regardless of length of time on dialysis, is in need of comprehensive professional oral care and self-care instruction. Oral disease was present and is a source of active infection in these medically compromised individuals and, as such, has dire implication for morbidity and mortality.